JNK is constitutively active in mantle cell lymphoma: cell cycle deregulation and polyploidy by JNK inhibitor SP600125

Mantle cell lymphoma (MCL) is characterized by genetic instability and a poor prognosis. Many blastoid variants are (hypo)tetraploid and have an even worse prognosis. We investigated the role of signalling by mitogen‐activated protein kinases (MAPKs) in MCL. As compared to normal tonsil B cells, MCL cells showed higher activation of the JNK MAPK in both an MAPK array and a sandwich ELISA assay. Immunohistochemistry showed overexpression of phospho (p)‐JNK (Thr183/Tyr185) in 30 of 37 MCL cases. Inhibition of p‐JNK with SP600125 resulted in growth arrest in all four MCL cell lines (Jeko‐1, HBL‐2, UPN‐1, Granta‐519), which could be partly reversed by the addition of CD40L and IL‐4. Furthermore, SP600125 led to G2/M phase arrest on day 1 and a striking increase in endoreduplication on day 2 and day 3, which was confirmed by karyotype analysis. G2/M arrest was associated with down‐regulation of EGR1 and p21 protein expression. SP600125‐induced polyploidy could be blocked by the BCL‐2 inhibitor YC137. These data suggest that constitutive JNK activity is necessary to promote proliferation and maintain diploidy in MCL. JNK inhibition leads to cell cycle deregulation and endoreduplication, mimicking the tetraploid state seen in a subset of MCL cases. Thus, our data also provide an experimental model to study polyploid MCL cells. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

JNK信号通路在NaAsO2诱导L-02肝细胞 凋亡过程中的作用

摘要:目的　研究JNK在NaAsO2诱导L-02肝细胞凋亡中的作用。方法　用不同浓度NaAsO2染毒L-02肝细胞,采用流式细胞术检测L-02肝细胞凋亡率;Western-blot检测L-02肝细胞中Jnk、p-Jnk、Caspase-3蛋白的相对表达量。结果　0、50、100 和 150 μmol/L组细胞凋亡率分别为3.33%±0.30%、7.49%±0.23%、9.48%±0.49%和 14.87%±2.17%,染毒组的细胞凋亡率随NaAsO2浓度的增加而升高,差异有统计学意义（P＜0.05）;染毒组细胞内Jnk、p-Jnk、Caspase-3蛋白相对表达量均增高,差异有统计学意义（P＜0.05）。结论　NaAsO2能诱导L-02肝细胞凋亡的发生,这可能与激活JNK,增加JNK磷酸化水平,继而激活凋亡调控基因Caspase-3有关。     

c-Jun N-terminal kinase-mediated Rubicon expression enhances hepatocyte lipoapoptosis and promotes hepatocyte ballooning

AIM: To clarify the relationship between autophagy and lipotoxicity-induced apoptosis, which is termed "lipoapoptosis," in non-alcoholic steatohepatitis. METHODS: Male C57BL/6J mice were fed a high-fat diet(HFD) for 12 wk, after which the liver histology and expression of proteins such as p62 or LC3 were evaluated. Alpha mouse liver 12(AML12) cells treated with palmitate(PA) were used as an in vitro model. RESULTS: LC3-Ⅱ, p62, and Run domain Beclin-1 interacting and cysteine-rich containing(Rubicon) proteins increased in both the HFD mice and in AML12 cells in response to PA treatment. Rubicon expression was decreased upon c-Jun N-terminal kinase(JNK) inhibition at both the m RNA and the protein level in AML12 cells. Rubicon knockdown in AML12 cells with PA decreased the protein levels of both LC3-Ⅱ and p62. Rubicon expression peaked at 4 h of PA treatment in AML12, and then decreased. Treatment with caspase-9 inhibitor ameliorated the decrease in Rubicon protein expression at 10 h of PA and resulted in enlarged AML12 cells under PA treatment. The enlargement of AML12 cells by PA with caspase-9 inhibition was canceled by Rubicon knockdown.CONCLUSION: The JNK-Rubicon axis enhanced lipoapoptosis, and caspase-9 inhibition and Rubicon had effects that were cytologically similar to hepatocyte ballooning. As ballooned hepatocytes secrete fibrogenic signals and thus might promote fibrosis in the liver, the inhibition of hepatocyte ballooning might provide antifibrosis in the NASH liver.     

Platycodin D induces apoptosis and triggers ERK- and JNK-mediated autophagy in human hepatocellular carcinoma BEL-7402 cells

Aim:

Platycodin D, the main saponin isolated from Chinese herb Platycodonis Radix, exhibits anticancer activities against various cancer cell lines. Here we evaluated its anticancer action against human hepatocellular carcinoma cells in vitro and in vivo, and elucidated the relationship between platycodin D-induced apoptosis and autophagy.

Methods:

The viability of human hepatocellular carcinoma BEL-7402 cells was evaluated with MTT assay, and the apoptosis was examined using Annexin V/PI and Hoechst 33342 staining assays. Monodansylcadaverine (MDC) staining was used to label autophagic vacuoles. The proteins were detected using Western blot analysis. For studying its anticancer action in vivo, platycodin D (5 and 10 mg· kg⁻¹·d⁻¹) was intraperitoneally injected to BEL-7402-bearing mice for 21 days.

Results:

Platycodin D (5–40 μmol/L) inhibited the cell proliferation in vitro with IC50 values of 37.70±3.99, 24.30±2.30 and 19.70±2.36 μmol/L at 24, 48 and 72 h, respectively. Platycodin D (5–20 μmol/L) dose-dependently increased BEL-7402 cell apoptosis, increased the Bax/Bcl-2 ratio and the levels of cleaved PARP and cleaved caspase-3, and decreased the level of Bcl-2. Furthermore, platycodin D (5–20 μmol/L) induced autophagy in BEL-7402 cells, as evidenced by formation of cytoplasmic vacuoles, increased amounts of LC3-II, and increased numbers of MDC-positive cells. Pretreatment with the autophagy inhibitor chloroquine (5 μmol/L) or BAF (50 nmol/L) significantly enhanced platycodin D-induced proliferation inhibition and apoptosis. Moreover, platycodin D (20 μmol/L) activated the ERK and JNK pathways in BEL-7402 cells, and simultaneous blockage of the two pathways effectively suppressed platycodin D-induced autophagy and enhanced platycodin D-induced apoptosis. In BEL-7402-bearing mice, platycodin D (10 mg·kg⁻¹•d⁻¹) significantly reduced relative tumor volume with decreased body weight.

Conclusion:

Platycodin D not only inhibits the proliferation of BEL-7402 cells but also suppresses BEL-7402 xenograft tumor growth. Platycodin D-induced cell proliferation inhibition and apoptosis are amplified by co-treatment with autophagy inhibitors     

兔痉挛脑血管中JNK的表达及其抑制剂的影响

目的 观察兔脑血管痉挛（CVS）后基底动脉p-jnk的表达及其抑制剂SP600125对其表达的影响，以探讨蛛网膜下腔出血（SAH）后CVS细胞信号转导通路机制。方法 ①采用二次枕大池注血建立家兔CVS模型。②采用HE染色、免疫组织化学技术动态观察兔基底动脉血管壁组织病理改变、p-jnk的表达及使用SP600125干预后对上述表达的影响。结果 ①SAH组基底动脉管腔狭窄、炎症细胞浸润等，以SAH后7d时为最重，10d时逐渐缓解；SAH＋SP600125组与同时段的SAH组比较血管变化明显减轻。②SAH组p-jnk在SAH后7d时表达最为明显，10d表达稍减少，但仍明显高于正常组（P〈0．01）；SAH＋SP600125组与同时段的SAH组比较p-jnk表达明显减少（P〈0．05）。结论 ①原癌基因c-jun氨基末端激酶（JNK）信号转导通路调节免疫炎性反应，是参与形成CVS机制之一。②JNK抑制剂SP600125可以调控血管壁的炎症反应，有效缓解CVS。     

JNK信号通路介导化学性缺氧对PC12细胞的损伤作用

目的探讨C-Jun蛋白氨基末端激酶(C-Jun N-termi-nal kinase,JNK)通路在化学性缺氧模拟剂氯化钴(CoCl2)对PC12细胞损伤中的作用。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理PC12细胞以建立化学性缺氧损伤模型。应用CCK-8比色法检测细胞存活率;Hoechst33258核染色法观察细胞凋亡的形态学改变;JC-1染色荧光显微镜照像检测线粒体膜电位(MMP);Western blot法检测JNK蛋白的表达水平。结果 600μmol.L-1CoCl2作用PC12细胞不同时间(12～48 h)后,可时间依赖性地抑制PC12细胞的存活率;600μmol.L-1的CoCl2处理PC12细胞48 h时,可引起细胞出现核固缩等典型的凋亡特征;CoCl2能明显的降低PC12细胞的MMP;CoCl2能诱导JNK的磷酸化,特异性的JNK阻断剂SP600125可抑制CoCl2对PC12细胞的上述损伤作用。结论 CoCl2可引起PC12细胞损伤,此作用可能与其诱导JNK磷酸化有关。     

Role of c-Jun N-terminal kinase in early brain injury after subarachnoid hemorrhage

The c-Jun N-terminal kinase (JNK) is induced by cerebral ischemia and injurious blood components acutely after subarachnoid hemorrhage (SAH). We hypothesized that inhibition of JNK will prevent damage to the neurovascular unit in the early brain injury period after SAH. Ninety-nine male SD rats (300-350 g) were randomly assigned to sham, SAH, and SAH treated with JNK inhibitor groups. SAH was induced by endovascular perforation. The JNK inhibitor SP600125 was administered intraperitoneally at 1 hr before and 6 hr after SAH. At 24 hr after SAH, we observed increased phosphorylation of JNK and c-Jun. Signs of neurovascular damage were observed in the hemorrhagic brains; these included the increases of aquaporin (AQP)-1 expression and brain water content as well as enhanced matrix metalloproteinase (MMP)-9 activity, vascular collagen IV loss, increased VEGF tissue level, and Evans blue extravasation. The appearances of cleaved caspase-3 expression, TUNEL-positive cells, and apoptotic morphology in cerebral tissues were associated with neurological deficit after SAH. JNK inhibition prevented c-Jun phosphorylation and suppressed AQP1, MMP-9, VEGF, and caspase-3 activation, with concomitant diminution of neuronal injury, blood-brain barrier preservation, reduced brain swelling, and improved neurological deficit in rats after SAH. This study demonstrates a multitude of beneficial effects of JNK inhibition, including protection of the neurovascular unit in early brain injury after SAH.     

鞘内注射JNK特异性抑制剂SP600125对CCI大鼠痛行为的影响

Objective To observe the effect of intrathecal administration of SP600125 on both MWT and TWL of rats after chronic constriction injury (CCI) of the sciatic nerve. Methods 40 male SD rats were randomized to deride into 5 groups (n=8). Rats in group SP5 received SP600125 5 μg after CCI; rats in group SP25 received SP600125 25 μg after CCI; rats in group SP50 received SP600125 50 μg after CCI; rats in group DMSO received 2% DMSO 10 μl after CCI; rats in group Naive received SP600125 50 μg without sciatic nerve injury. SP600125 was dissolved in 10 μl 2%DMSO solvent. On the 7th day after CCI, MWT and TWL were determined with yon Frey filaments and thermal radiation apparatus repectively after intrathecal administration of SP600125. Results Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI rather than normal rats. Conclusion Intrathecal administration certain dosage of SP600125 could attenuate the established mechanical allodynia and thermal hyperalgesia induced by CCI.